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1.
J Mol Cell Cardiol ; 2021 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-34742715

RESUMO

RNA sequencing is a powerful tool to analyze gene expression transcriptome wide. However, RNA sequencing in general and especially the recently developed methods of long read RNA sequencing are still low-throughput and cost-intensive. Here, one important design choice is to concentrate the sequencing capacity on specific parts of the transcriptome. Especially, abundant transcripts as ribosomal RNAs may dominate the available sequencing space, if not removed prior to sequencing. Several methods exist to reduce ribosomal RNA read numbers: either based on enrichment of the relevant fraction (polyA+ RNA) or depletion, respectively degradation of ribosomal RNAs. Furthermore, commercial kits are available to deplete globin transcripts from blood samples. However, so far, no solution exists to deal with other tissue-specific highly abundant transcripts. This is especially of interest in the heart and other muscle derived samples, where reads originating from mitochondrial RNAs make up to 30% of reads in polyA+ selected libraries and around 70% in single cell sequencing experiments. We present a simple method to diminish sequencing of mitochondrial RNAs in Oxford Nanopore direct RNA sequencing libraries by RNase H based clipping of the polyA tail. We show that mt-clipping enables enhanced detection of cytoplasmic mRNAs, among them genes involved in heart development and pathogenesis. Mt-clipping may be applied as well to other sequencing protocols that are based on oligo(dT) priming and can be easily adapted to other tissue-specific high-abundant transcripts.

2.
Circ Res ; 2021 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-34789007

RESUMO

Background: Circular RNAs (circRNAs) are generated by back-splicing of mostly mRNAs and are gaining increasing attention as a novel class of regulatory RNAs that control various cellular functions. However, their physiological roles and functional conservation in vivo are rarely addressed, given the inherent challenges of their genetic inactivation. Here we aimed to identify locus conserved circRNAs in mice and humans, which can be genetically deleted due to retained intronic elements not contained in the mRNA host gene to eventually address functional conservation. Methods: Mechanistically, we identified the protein syndesmos (SDOS) to specifically interact with cZNF292 in endothelial cells by RNA affinity purification and subsequent mass spectrometry analysis. Silencing of SDOS or its protein binding partner Syndecan-4, or mutation of the SDOS-cZNF292 binding site, prevented laminar flow-induced cytoskeletal reorganisation thereby recapitulating cZfp292 phenotypes. Results: Combining published endothelial RNA sequencing datasets with circRNAs of the circATLAS databank, we identified locus-conserved circRNA retaining intronic elements between mice and humans. CRISPR/Cas9 mediated genetic depletion of the top expressed circRNA cZfp292 resulted in an altered endothelial morphology and aberrant flow alignment in the aorta in vivo. Consistently, depletion of cZNF292 in endothelial cells in vitro abolished laminar flow-induced alterations in cell orientation, paxillin localisation and focal adhesion organisation. Conclusion: Together, our data reveal a hitherto unknown role of cZNF292/cZfp292 in endothelial flow responses, which influences endothelial shape.

3.
EMBO Rep ; : e52170, 2021 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-34605609

RESUMO

The mechanistic target of rapamycin (mTOR) promotes pathological remodeling in the heart by activating ribosomal biogenesis and mRNA translation. Inhibition of mTOR in cardiomyocytes is protective; however, a detailed role of mTOR in translational regulation of specific mRNA networks in the diseased heart is unknown. We performed cardiomyocyte genome-wide sequencing to define mTOR-dependent gene expression control at the level of mRNA translation. We identify the muscle-specific protein Cullin-associated NEDD8-dissociated protein 2 (Cand2) as a translationally upregulated gene, dependent on the activity of mTOR. Deletion of Cand2 protects the myocardium against pathological remodeling. Mechanistically, we show that Cand2 links mTOR signaling to pathological cell growth by increasing Grk5 protein expression. Our data suggest that cell-type-specific targeting of mTOR might have therapeutic value against pathological cardiac remodeling.

4.
Nat Commun ; 12(1): 5864, 2021 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-34620876

RESUMO

Pausing of RNA polymerase II (Pol II) close to promoters is a common regulatory step in RNA synthesis, and is coordinated by a ribonucleoprotein complex scaffolded by the noncoding RNA RN7SK. The function of RN7SK-regulated gene transcription in adult tissue homoeostasis is currently unknown. Here, we deplete RN7SK during mouse and human epidermal stem cell differentiation. Unexpectedly, loss of this small nuclear RNA specifically reduces transcription of numerous cell cycle regulators leading to cell cycle exit and differentiation. Mechanistically, we show that RN7SK is required for efficient transcription of highly expressed gene pairs with bidirectional promoters, which in the epidermis co-regulated cell cycle and chromosome organization. The reduction in transcription involves impaired splicing and RNA decay, but occurs in the absence of chromatin remodelling at promoters and putative enhancers. Thus, RN7SK is directly required for efficient Pol II transcription of highly transcribed bidirectional gene pairs, and thereby exerts tissue-specific functions, such as maintaining a cycling cell population in the epidermis.


Assuntos
Regulação da Expressão Gênica , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Pele/metabolismo , Transcrição Genética , Animais , Ciclo Celular , Diferenciação Celular , Proliferação de Células , Cromatina , Montagem e Desmontagem da Cromatina , Epiderme , Feminino , Humanos , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Splicing de RNA , Pele/patologia , Células-Tronco
5.
Stud Health Technol Inform ; 283: 146-155, 2021 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-34545830

RESUMO

Wearables are commercially available devices allowing continuous monitoring of users' health parameters. Their easy availability, increasing accuracy and functionality render them relevant for medical practice, specifically for longitudinal monitoring. There are clear benefits for the health care system, such as the opportunity of timely interventions by monitoring a patient during his daily life, resulting in a cost reduction in medical care and improved patient well-being. However, some tools are essential to enable the application of wearables in medical daily practice. For example, there is a need for software solutions that allow clinicians to quickly and easily analyze data from devices of their patients. The goal of this study was to develop a dashboard for physicians, which allows rapid data interpretation of longitudinal data from the Apple Watch. The prototype dashboard is an interactive web-based visualization platform utilizing Plotly. The dashboard displays the most important parameters like heart rate, steps per day, activity, exercise collected by the Apple Watch in a user-friendly and accessible way. Clear visualization makes it easy to identify trends or deviations in the data and see how these changes in daily behaviour affect patients' health. Our software is a key component to monitor patients with heart failure who participate in the HiGHmed use case cardiology project.


Assuntos
Exercício Físico , Insuficiência Cardíaca , Insuficiência Cardíaca/terapia , Frequência Cardíaca , Humanos , Monitorização Fisiológica , Software
6.
EMBO Rep ; 22(10): e52094, 2021 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-34396684

RESUMO

Synaptic scaling is a form of homeostatic plasticity which allows neurons to adjust their action potential firing rate in response to chronic alterations in neural activity. Synaptic scaling requires profound changes in gene expression, but the relative contribution of local and cell-wide mechanisms is controversial. Here we perform a comprehensive multi-omics characterization of the somatic and process compartments of primary rat hippocampal neurons during synaptic scaling. We uncover both highly compartment-specific and correlating changes in the neuronal transcriptome and proteome. Whereas downregulation of crucial regulators of neuronal excitability occurs primarily in the somatic compartment, structural components of excitatory postsynapses are mostly downregulated in processes. Local inhibition of protein synthesis in processes during scaling is confirmed for candidate synaptic proteins. Motif analysis further suggests an important role for trans-acting post-transcriptional regulators, including RNA-binding proteins and microRNAs, in the local regulation of the corresponding mRNAs. Altogether, our study indicates that, during synaptic scaling, compartmentalized gene expression changes might co-exist with neuron-wide mechanisms to allow synaptic computation and homeostasis.


Assuntos
Plasticidade Neuronal , Sinapses , Animais , Expressão Gênica , Regulação da Expressão Gênica , Plasticidade Neuronal/genética , Neurônios , Ratos
8.
Brief Bioinform ; 22(6)2021 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-34228787

RESUMO

Metabolic labeling of newly transcribed RNAs coupled with RNA-seq is being increasingly used for genome-wide analysis of RNA dynamics. Methods including standard biochemical enrichment and recent nucleotide conversion protocols each require special experimental and computational treatment. Despite their immediate relevance, these technologies have not yet been assessed and benchmarked, and no data are currently available to advance reproducible research and the development of better inference tools. Here, we present a systematic evaluation and comparison of four RNA labeling protocols: 4sU-tagging biochemical enrichment, including spike-in RNA controls, SLAM-seq, TimeLapse-seq and TUC-seq. All protocols are evaluated based on practical considerations, conversion efficiency and wet lab requirements to handle hazardous substances. We also compute decay rate estimates and confidence intervals for each protocol using two alternative statistical frameworks, pulseR and GRAND-SLAM, for over 11 600 human genes and evaluate the underlying computational workflows for their robustness and ease of use. Overall, we demonstrate a high inter-method reliability across eight use case scenarios. Our results and data will facilitate reproducible research and serve as a resource contributing to a fuller understanding of RNA biology.

9.
Basic Res Cardiol ; 116(1): 42, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34224022

RESUMO

Coronavirus disease 2019 (COVID-19) spawned a global health crisis in late 2019 and is caused by the novel coronavirus SARS-CoV-2. SARS-CoV-2 infection can lead to elevated markers of endothelial dysfunction associated with higher risk of mortality. It is unclear whether endothelial dysfunction is caused by direct infection of endothelial cells or is mainly secondary to inflammation. Here, we investigate whether different types of endothelial cells are susceptible to SARS-CoV-2. Human endothelial cells from different vascular beds including umbilical vein endothelial cells, coronary artery endothelial cells (HCAEC), cardiac and lung microvascular endothelial cells, or pulmonary arterial cells were inoculated in vitro with SARS-CoV-2. Viral spike protein was only detected in HCAECs after SARS-CoV-2 infection but not in the other endothelial cells tested. Consistently, only HCAEC expressed the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2), required for virus infection. Infection with the SARS-CoV-2 variants B.1.1.7, B.1.351, and P.2 resulted in significantly higher levels of viral spike protein. Despite this, no intracellular double-stranded viral RNA was detected and the supernatant did not contain infectious virus. Analysis of the cellular distribution of the spike protein revealed that it co-localized with endosomal calnexin. SARS-CoV-2 infection did induce the ER stress gene EDEM1, which is responsible for clearance of misfolded proteins from the ER. Whereas the wild type of SARS-CoV-2 did not induce cytotoxic or pro-inflammatory effects, the variant B.1.1.7 reduced the HCAEC cell number. Of the different tested endothelial cells, HCAECs showed highest viral uptake but did not promote virus replication. Effects on cell number were only observed after infection with the variant B.1.1.7, suggesting that endothelial protection may be particularly important in patients infected with this variant.


Assuntos
Retículo Endoplasmático/virologia , Células Endoteliais/virologia , SARS-CoV-2/patogenicidade , Enzima de Conversão de Angiotensina 2/metabolismo , Calnexina/metabolismo , Células Cultivadas , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Células Endoteliais/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Proteínas de Membrana/metabolismo , Receptores Virais/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo
10.
Nat Commun ; 12(1): 3965, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34172724

RESUMO

Eukaryotic gene expression is constantly controlled by the translation-coupled nonsense-mediated mRNA decay (NMD) pathway. Aberrant translation termination leads to NMD activation, resulting in phosphorylation of the central NMD factor UPF1 and robust clearance of NMD targets via two seemingly independent and redundant mRNA degradation branches. Here, we uncover that the loss of the first SMG5-SMG7-dependent pathway also inactivates the second SMG6-dependent branch, indicating an unexpected functional connection between the final NMD steps. Transcriptome-wide analyses of SMG5-SMG7-depleted cells confirm exhaustive NMD inhibition resulting in massive transcriptomic alterations. Intriguingly, we find that the functionally underestimated SMG5 can substitute the role of SMG7 and individually activate NMD. Furthermore, the presence of either SMG5 or SMG7 is sufficient to support SMG6-mediated endonucleolysis of NMD targets. Our data support an improved model for NMD execution that features two-factor authentication involving UPF1 phosphorylation and SMG5-SMG7 recruitment to access SMG6 activity.


Assuntos
Proteínas de Transporte/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido/fisiologia , Proteínas de Transporte/química , Proteínas de Transporte/genética , Linhagem Celular , Feminino , Técnicas de Inativação de Genes , Humanos , Fosforilação , RNA Helicases/genética , RNA Helicases/metabolismo , Telomerase/metabolismo , Transativadores/genética , Transativadores/metabolismo
11.
Elife ; 102021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34152268

RESUMO

In the adult heart, the epicardium becomes activated after injury, contributing to cardiac healing by secretion of paracrine factors. Here, we analyzed by single-cell RNA sequencing combined with RNA in situ hybridization and lineage tracing of Wilms tumor protein 1-positive (WT1+) cells, the cellular composition, location, and hierarchy of epicardial stromal cells (EpiSC) in comparison to activated myocardial fibroblasts/stromal cells in infarcted mouse hearts. We identified 11 transcriptionally distinct EpiSC populations, which can be classified into three groups, each containing a cluster of proliferating cells. Two groups expressed cardiac specification markers and sarcomeric proteins suggestive of cardiomyogenic potential. Transcripts of hypoxia-inducible factor (HIF)-1α and HIF-responsive genes were enriched in EpiSC consistent with an epicardial hypoxic niche. Expression of paracrine factors was not limited to WT1+ cells but was a general feature of activated cardiac stromal cells. Our findings provide the cellular framework by which myocardial ischemia may trigger in EpiSC the formation of cardioprotective/regenerative responses.


Assuntos
Fibroblastos/metabolismo , Miocárdio/metabolismo , Pericárdio/fisiologia , Células Estromais/metabolismo , Transcriptoma , Animais , Perfilação da Expressão Gênica , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA , Análise de Sequência de RNA , Análise de Célula Única , Proteínas WT1/metabolismo
12.
Nucleic Acids Res ; 49(16): e92, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34157120

RESUMO

N6-methyladenosine (m6A) is the most abundant internal RNA modification in eukaryotic mRNAs and influences many aspects of RNA processing. miCLIP (m6A individual-nucleotide resolution UV crosslinking and immunoprecipitation) is an antibody-based approach to map m6A sites with single-nucleotide resolution. However, due to broad antibody reactivity, reliable identification of m6A sites from miCLIP data remains challenging. Here, we present miCLIP2 in combination with machine learning to significantly improve m6A detection. The optimized miCLIP2 results in high-complexity libraries from less input material. Importantly, we established a robust computational pipeline to tackle the inherent issue of false positives in antibody-based m6A detection. The analyses were calibrated with Mettl3 knockout cells to learn the characteristics of m6A deposition, including m6A sites outside of DRACH motifs. To make our results universally applicable, we trained a machine learning model, m6Aboost, based on the experimental and RNA sequence features. Importantly, m6Aboost allows prediction of genuine m6A sites in miCLIP2 data without filtering for DRACH motifs or the need for Mettl3 depletion. Using m6Aboost, we identify thousands of high-confidence m6A sites in different murine and human cell lines, which provide a rich resource for future analysis. Collectively, our combined experimental and computational methodology greatly improves m6A identification.


Assuntos
Adenosina/análogos & derivados , Aprendizado de Máquina , Processamento Pós-Transcricional do RNA , RNA-Seq/métodos , Adenosina/química , Adenosina/metabolismo , Animais , Células HEK293 , Humanos , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Motivos de Nucleotídeos , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA-Seq/normas , Sensibilidade e Especificidade
13.
Stud Health Technol Inform ; 278: 187-194, 2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34042893

RESUMO

The HiGHmed consortium aims to create a shared information governance framework to integrate clinical routine data. One challenge is the replacement of unstructured reporting (e.g. doctoral letters) with structured reporting in clinical routine. The Heidelberg cardiology department evaluates dynamic PDF forms for structured data reporting of heart failure (HF) patients. In this use case, we aim to identify potential caveats or shortcomings in data processing at an early stage. We employed data mining strategies to detect patterns related to incomplete or false data, which we found to be present among all data types. We then discuss the characteristics of the baseline patient cohort in Heidelberg to find out about specific peculiarities and potential biases, which may be site-specific. Briefly, our patient population is predominantly male (67%), NYHA I & II are the most common severity classes, NYHA IV is missing entirely. Most patients have a dilated cardiomyopathy (DCM) or coronary heart disease (CHD) diagnosed as their cause of HF. Finally, we also analyzed how comorbidities and risk factors relate to specific disease entities of heart failure patients. Family anamnesis was more frequent among cardiomyopathy patients than among CHD patients, who show a more dominating presence of dyslipidemia instead. Generally, the most dominant risk factor was arterial hypertension, while at the other end of the scale alcoholism appears to be underreported.


Assuntos
Cardiologia , Insuficiência Cardíaca , Estudos de Coortes , Insuficiência Cardíaca/epidemiologia , Humanos , Masculino , Fatores de Risco
14.
RNA ; 2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33906975

RESUMO

The current ecosystem of single cell RNA-seq platforms is rapidly expanding, but robust solutions for single cell and single molecule full- length RNA sequencing are virtually absent. A high-throughput solution that covers all aspects is necessary to study the complex life of mRNA on the single cell level. The Nanopore platform offers long read sequencing and can be integrated with the popular single cell sequencing method on the 10x Chromium platform. However, the high error-rate of Nanopore reads poses a challenge in downstream processing (e.g. for cell barcode assignment). We propose a solution to this particular problem by using a hybrid sequencing approach on Nanopore and Illumina platforms. Our software ScNapBar enables cell barcode assignment with high accuracy, especially if sequencing satura- tion is low. ScNapBar uses unique molecular identifier (UMI) or Naive Bayes probabilistic approaches in the barcode assignment, depending on the available Illumina sequencing depth. We have benchmarked the two approaches on simulated and real Nanopore datasets. We further applied ScNapBar to pools of cells with an active or a silenced non-sense mediated RNA decay pathway. Our Nanopore read assignment distinguishes the respective cell populations and reveals characteristic nonsense-mediated mRNA decay events depending on cell status.

15.
Cells ; 10(3)2021 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-33804736

RESUMO

Diseases of the renal filtration unit-the glomerulus-are the most common cause of chronic kidney disease. Podocytes are the pivotal cell type for the function of this filter and focal-segmental glomerulosclerosis (FSGS) is a classic example of a podocytopathy leading to proteinuria and glomerular scarring. Currently, no targeted treatment of FSGS is available. This lack of therapeutic strategies is explained by a limited understanding of the defects in podocyte cell biology leading to FSGS. To date, most studies in the field have focused on protein-coding genes and their gene products. However, more than 80% of all transcripts produced by mammalian cells are actually non-coding. Here, long non-coding RNAs (lncRNAs) are a relatively novel class of transcripts and have not been systematically studied in FSGS to date. The appropriate tools to facilitate lncRNA research for the renal scientific community are urgently required due to a row of challenges compared to classical analysis pipelines optimized for coding RNA expression analysis. Here, we present the bioinformatic pipeline CALINCA as a solution for this problem. CALINCA automatically analyzes datasets from murine FSGS models and quantifies both annotated and de novo assembled lncRNAs. In addition, the tool provides in-depth information on podocyte specificity of these lncRNAs, as well as evolutionary conservation and expression in human datasets making this pipeline a crucial basis to lncRNA studies in FSGS.


Assuntos
Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/patologia , Podócitos/metabolismo , Podócitos/patologia , RNA Longo não Codificante/metabolismo , Software , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos Endogâmicos BALB C , RNA Longo não Codificante/genética , Reprodutibilidade dos Testes
16.
Methods Mol Biol ; 2252: 295-312, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33765282

RESUMO

Ribosome profiling has been instrumental in leading to important discoveries in several fields of life sciences. Here we describe a computational approach that enables identification of translation events on a genome-wide scale from ribosome profiling data. Periodic fragment sizes indicative of active translation are selected without supervision for each library. Our workflow allows to map the whole translational landscape of a given cell, tissue, or organism, under varying conditions, and can be used to expand the search for novel, uncharacterized open reading frames, such as regulatory upstream translation events. Through a detailed workflow example, we show how to perform qualitative and quantitative analysis of translatomes.


Assuntos
Biologia Computacional/métodos , RNA Mensageiro/genética , Ribossomos/metabolismo , Teorema de Bayes , Humanos , Fases de Leitura Aberta , Biossíntese de Proteínas , Análise de Sequência de RNA , Software , Aprendizado de Máquina não Supervisionado , Fluxo de Trabalho
17.
Nat Commun ; 12(1): 463, 2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33469025

RESUMO

Splicing varies across brain regions, but the single-cell resolution of regional variation is unclear. We present a single-cell investigation of differential isoform expression (DIE) between brain regions using single-cell long-read sequencing in mouse hippocampus and prefrontal cortex in 45 cell types at postnatal day 7 ( www.isoformAtlas.com ). Isoform tests for DIE show better performance than exon tests. We detect hundreds of DIE events traceable to cell types, often corresponding to functionally distinct protein isoforms. Mostly, one cell type is responsible for brain-region specific DIE. However, for fewer genes, multiple cell types influence DIE. Thus, regional identity can, although rarely, override cell-type specificity. Cell types indigenous to one anatomic structure display distinctive DIE, e.g. the choroid plexus epithelium manifests distinct transcription-start-site usage. Spatial transcriptomics and long-read sequencing yield a spatially resolved splicing map. Our methods quantify isoform expression with cell-type and spatial resolution and it contributes to further our understanding of how the brain integrates molecular and cellular complexity.


Assuntos
Processamento Alternativo/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hipocampo/metabolismo , Córtex Pré-Frontal/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Animais Recém-Nascidos , Biologia Computacional , Feminino , Hipocampo/citologia , Hipocampo/crescimento & desenvolvimento , Camundongos , Modelos Animais , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/crescimento & desenvolvimento , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Análise de Célula Única/métodos , Análise Espacial
18.
EMBO J ; 40(4): e104975, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33428246

RESUMO

N6-methyladenosine (m6 A) regulates a variety of physiological processes through modulation of RNA metabolism. This modification is particularly enriched in the nervous system of several species, and its dysregulation has been associated with neurodevelopmental defects and neural dysfunctions. In Drosophila, loss of m6 A alters fly behavior, albeit the underlying molecular mechanism and the role of m6 A during nervous system development have remained elusive. Here we find that impairment of the m6 A pathway leads to axonal overgrowth and misguidance at larval neuromuscular junctions as well as in the adult mushroom bodies. We identify Ythdf as the main m6 A reader in the nervous system, being required to limit axonal growth. Mechanistically, we show that the m6 A reader Ythdf directly interacts with Fmr1, the fly homolog of Fragile X mental retardation RNA binding protein (FMRP), to inhibit the translation of key transcripts involved in axonal growth regulation. Altogether, this study demonstrates that the m6 A pathway controls development of the nervous system and modulates Fmr1 target transcript selection.


Assuntos
Adenosina/análogos & derivados , Axônios/fisiologia , Proteínas de Drosophila/metabolismo , Proteína do X Frágil de Retardo Mental/metabolismo , Neurônios/citologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adenosina/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteína do X Frágil de Retardo Mental/genética , Neurônios/fisiologia , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética
19.
J Mol Cell Cardiol ; 150: 23-31, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33049256

RESUMO

Clinically translatable large animal models have become indispensable for cardiovascular research, clinically relevant proof of concept studies and for novel therapeutic interventions. In particular, the pig has emerged as an essential cardiovascular disease model, because its heart, circulatory system, and blood supply are anatomically and functionally similar to that of humans. Currently, molecular and omics-based studies in the pig are hampered by the incompleteness of the genome and the lack of diversity of the corresponding transcriptome annotation. Here, we employed Nanopore long-read sequencing and in-depth proteomics on top of Illumina RNA-seq to enhance the pig cardiac transcriptome annotation. We assembled 15,926 transcripts, stratified into coding and non-coding, and validated our results by complementary mass spectrometry. A manual review of several gene loci, which are associated with cardiac function, corroborated the utility of our enhanced annotation. All our data are available for download and are provided as tracks for integration in genome browsers. We deem this resource as highly valuable for molecular research in an increasingly relevant large animal model.


Assuntos
Miocárdio/metabolismo , Proteômica , Análise de Sequência de RNA , Suínos/genética , Transcriptoma/genética , Animais , Anotação de Sequência Molecular , Sequenciamento por Nanoporos , Fases de Leitura Aberta/genética
20.
Prog Biophys Mol Biol ; 159: 86-104, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32738354

RESUMO

AIMS: After summarizing current concepts for the role of TRPC cation channels in cardiac cells and in processes triggered by mechanical stimuli arising e.g. during pressure overload, we analysed the role of TRPC1 and TRPC4 for background Ca2+ entry (BGCE) and for cardiac pressure overload induced transcriptional remodelling. METHODS AND RESULTS: Mn2+-quench analysis in cardiomyocytes from several Trpc-deficient mice revealed that both TRPC1 and TRPC4 are required for BGCE. Electrically-evoked cell shortening of cardiomyocytes from TRPC1/C4-DKO mice was reduced, whereas parameters of cardiac contractility and relaxation assessed in vivo were unaltered. As pathological cardiac remodelling in mice depends on their genetic background, and the development of cardiac remodelling was found to be reduced in TRPC1/C4-DKO mice on a mixed genetic background, we studied TRPC1/C4-DKO mice on a C57BL6/N genetic background. Cardiac hypertrophy was reduced in those mice after chronic isoproterenol infusion (-51.4%) or after one week of transverse aortic constriction (TAC; -73.0%). This last manoeuvre was preceded by changes in the pressure overload induced transcriptional program as analysed by RNA sequencing. Genes encoding specific collagens, the Mef2 target myomaxin and the gene encoding the mechanosensitive channel Piezo2 were up-regulated after TAC in wild type but not in TRPC1/C4-DKO hearts. CONCLUSIONS: Deletion of the TRPC1 and TRPC4 channel proteins protects against development of pathological cardiac hypertrophy independently of the genetic background. To determine if the TRPC1/C4-dependent changes in the pressure overload induced alterations in the transcriptional program causally contribute to cardio-protection needs to be elaborated in future studies.


Assuntos
Cálcio/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Cátion TRPC/metabolismo , Remodelação Ventricular/fisiologia , Animais , Fenômenos Biomecânicos/fisiologia , Sinalização do Cálcio , Cardiomegalia/metabolismo , Regulação da Expressão Gênica , Humanos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Ativação Transcricional/fisiologia
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